Transmembrane proteins (TMPs) comprise more than 25% of the protein-coding potential of most genomes. They also play central roles in cell and organismal physiology and are the targets of a large fraction of all clinically useful drugs. However, there is a huge deficit in our knowledge of the structure and function of TMPs in comparison to soluble proteins. This can be primarily attributed to the substantial roadblocks generally encountered in applying x-ray crystallography to TMPs. This application brings together three independent PIs with diverse backgrounds in the molecular biology, biochemistry, biophysics, and structural biology, specifically of membrane proteins, to create a pipeline for TMP structure determination. A central tenet of the pipeline is the need to discriminate at the earliest possible stage in production between protein targets that are amenable to structure determination and those that are not. With this in mind, we propose to target families of orthologous, paralogous, and mutated proteins, carrying multiple variants through the early stages of purification and characterization so as to maximize the chances of advancing the most tractable members of a target family to the point of successful crystallization and diffraction. The project makes use of existing cloning and expression protocols for the bacterial and yeast expression systems that are most amenable to parallel expression strategies, but will use bacculovirus expression for some proteins. Expression testing will be conducted using small-scale growths; multiple forms of a given target will be produced at an intermediate scale to allow characterization using an existing high-throughput screen for detergent compatibility, biophysical and biochemical characterization and small-scale exploratory crystallization trials. Only the most promising candidates from intermediate scale analysis will be carried forward to large scale production for high-throughput crystallization screening using the facilities of the Hauptman-Woodward Institute. Additional screening using lipidic cubic phases will be conducted as needed. In addition to proteins expected from the PSI Network, initial structure determination efforts will target three classes of proteins: certain classes of transmembrane transporters, enzymes involved in lipid synthesis and lipid attachment to proteins, and complexes of seven-transmembrane segment proteins, including GPCRs, with single pass chaperone-like accessory proteins. The project also seeks to develop improved technologies for increasing levels of expression of functional TMPs in yeast, new approaches for specific fluorescent labeling of unpurified proteins, and the development of improved methods for biophysical characterization and screening of protein detergent complexes.